Volume 16, Issue 6, Pages 644-656 (June 2009) Amitriptyline is a TrkA and TrkB Receptor Agonist that Promotes TrkA/TrkB Heterodimerization and Has Potent Neurotrophic Activity  Sung-Wuk Jang, Xia Liu, Chi-Bun Chan, David Weinshenker, Randy A. Hall, Ge Xiao, Keqiang Ye  Chemistry & Biology  Volume 16, Issue 6, Pages 644-656 (June 2009) DOI: 10.1016/j.chembiol.2009.05.010 Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 1 Amitriptyline Selectively Protects Hippocampal Neurons from Apoptosis (A) Chemical Structures of tricyclic antidepressant drugs. (B) Some of the tricyclic antidepressant drugs protect T17-TrkA cells but not parental SN56 cells from apoptosis. (C) EC50 titration assays for promoting T17 cell survival. TrkA-overexpressing T17 cells were pretreated with various tricyclic antidepressant drugs for 30 min, followed by 1 μM staurosporine for 9 hr. Apoptosis was quantitatively analyzed. EC50 values are the drug concentrations, which prevent 50% of cells from apoptosis. (D) Amitriptyline selectively prevents apoptosis in hippocampal neurons. Hippocampal neurons were pretreated with NGF (100 ng/ml), gambogic amide, and various tricyclic antidepressant drugs (0.5 μM) for 30 min, followed by 50 μM glutamate for 16 hr. Apoptosis was quantitatively analyzed. (E) Amitriptyline prevents OGD-provoked neuronal apoptosis in hippocampal neurons. Hippocampal neurons were pretreated with various drugs (0.5 μM) for 30 min, followed by OGD for 3 hr. Apoptosis was quantitatively analyzed (left). Data represent the mean ± SEM of n = 4–5; one-way ANOVA, followed by Dunnett's test, ∗p < 0.01; ∗∗p < 0.005. Chemistry & Biology 2009 16, 644-656DOI: (10.1016/j.chembiol.2009.05.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 2 Amitriptyline Activates the TrkA Receptor and its Downstream Signaling Cascades (A) Amitriptyline activates TrkA in hippocampal neurons. Hippocampal neurons were treated with NGF (100 ng/ml) or various tricyclic antidepressant drugs (500 nM) for 30 min. Immunofluorescent staining was conducted with anti-p-TrkA antibody. (B) Amitriptyline activates TrkA downstream signaling cascades including Akt and MAP kinases. Hippocampal neurons were treated as described above. The cell lysates were analyzed by immunoblotting with various antibodies. (C) Trk receptor inhibitor K252a (100 nM) blocks amitriptyline-provoked TrkA and TrkB activation. Data represent mean ± SEM (One-way ANOVA, Dunnett's test, ∗p < 0.001). (D) Akt and MAP kinase activation kinetics and dosage assays by amitriptyline. Hippocampal neurons were treated with 500 nM amitriptyline for various time points. The cell lysates were analyzed by immunoblotting with various antibodies. Chemistry & Biology 2009 16, 644-656DOI: (10.1016/j.chembiol.2009.05.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 3 Amitriptyline Provokes Neurite Outgrowth in PC12 Cells (A) Amitriptyline but not other tricyclic compounds provokes neurite outgrowth in PC12 cells. PC12 cells were treated with amitriptyline and other compounds (500 nM) for 5 days in 2% FBS and 1% HS medium. The drug-containing medium was replenished every other day. Amitriptyline induced neurite outgrowth as potently as NGF (top). The relative neurite length was quantified (bottom). (B) Dose-dependent effect of neurite outgrowth. Amitriptyline (100 nM) was able to provoke neurite outgrowth in PC12 cells (One-way ANOVA, Bonferroni post hoc test, ∗p < 0.01; ∗∗p < 0.005). (C) K252a, PI3K, and MAP kinase inhibitors abolish amitriptyline-provoked neurite outgrowth in PC12 cells. Chemistry & Biology 2009 16, 644-656DOI: (10.1016/j.chembiol.2009.05.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 4 Amitriptyline Binds TrkA and TrkB Receptors and Triggers their Dimerization and Autophosphorylation (A) In vitro binding assay with Trk recombinant proteins. [3H]Amitriptyline (0.1 μCi) was incubated with purified ECD and ICD recombinant proteins of Trk receptors (5 μg each) at 30°C for 1 hr. The mixture was subjected to filter/vacuum assay. After extensive washing, the filter paper was analyzed in liquid scintillation counter. (B) Quantitative analysis of the binding between amitriptyline and TrkA and TrkB ECD. TrkA and TrkB ECD binding curve by [3H]amitriptyline (top). Scatchard plot for Kd analysis (bottom left). Cold aminitryptyline competed with [3H]amitriptyline for binding to TrkA or TrkB ECD (bottom right). (C) ECD domain in TrkA is essential for amitriptyline to bind TrkA receptor. (D) Amitriptyline associates with the first LRM in the N terminus of ECD of TrkA. Protein sequence alignment between the LRM motif from TrkA and the counterparts from TrkB and TrkC (top). In vitro binding assay was conducted with a variety of fragments of TrkA ECD recombinant proteins (bottom). Data represent mean ± SEM. (E) Amitriptyline provokes TrkA dimerization. GFP-TrkA and HA-TrkA or HA-TrkB were respectively cotransfected into HEK293 cells and treated with 0.5 μM amitriptyline or imipramine for 30 min. GFP-TrkA was immunoprecipitated with anti-GFP antibody, and the coprecipitated proteins were analyzed with anti-HA antibody. (F) Amitriptyline-triggered TrkA or TrkB tyrosine phosphorylation in HEK293 cells individually transfected by TrkA or TrkB, respectively. Kinase-dead TrkA displayed negligible tyrosine phosphorylation. Chemistry & Biology 2009 16, 644-656DOI: (10.1016/j.chembiol.2009.05.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 5 Amitriptyline Activates TrkA and TrkB Receptors and Prevents KA-Triggered Neuronal Apoptosis in Mouse Brain (A) Amitriptyline but not imipramine activates TrkA and TrkB receptors in mouse brain. Two- to three-month-old C57BL/6 mice were intraperitoneally injected with 15 mg/kg amitriptyline and 20 mg/kg imipramine for various time points. Immunoblotting was conducted with various indicated antibodies. (B) Amitriptyline and imipramine do not alter TrkA or TrkB transcription. RT-PCR analysis of TrkA and TrkB in mouse brain. (C) Amitriptyline diminishes KA-triggered hippocampal neuronal cell death. Two- to three-month-old C57BL/6 mice were intraperitoneally injected with 15 mg/kg amitriptyline either before or after KA (25 mg/kg) administration. In 5 days, the brain slides were analyzed with TUNEL assay. Green shows apoptotic nuclei, which were also stained with DAPI. (D) Amitriptyline increases TrkA expression and provokes TrkA and TrkB activation in hippocampus. Data represent mean ± SEM (n = 3/group, One-way ANOVA, Bonferroni post hoc test, ∗p < 0.01; ∗∗p < 0.01). Chemistry & Biology 2009 16, 644-656DOI: (10.1016/j.chembiol.2009.05.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 6 Amitriptyline Promotes TrkA and TrkB Heterodimerization in Mouse Brain (A) Amitriptyline provokes TrkA phosphorylation and upregulates its expression mice. Two- to three-month-old mice were intraperitoneally injected with various antidepressant drugs for 5 days. The doses were 15 mg/kg, 20 mg/kg, and 25 mg/kg for amitriptyline, imipramine, and fluoxetine, respectively. (B) Amitriptyline triggers TrkA and TrkB receptor heterodimerization in mouse brain. Wild-type and 5-HT1a null mice were treated with various antidepressant drugs for 5 days. TrkA was immunoprecipitated, and its coprecipitated proteins were analyzed by immunoblotting. (C) Amitriptyline elicits TrkA and TrkB receptor homo- and heterodimerization. Differentially tagged TrkA and TrkB receptors were cotransfected into HEK293 cells and treated with various antidepressant drugs for 30 min. Data represent mean ± SEM of n = 3. (D) Amitriptyline binding motif on TrkA is essential for Trk receptor homo- and heterodimerization. HA-tagged TrkA or TrkB was cotransfected with GST-ΔTrkA or GST-ΔTrkB into HEK293 cells, followed by NGF, BDNF, amitriptyline, or imipramine treatment. Truncated TrkA or TrkB was pulled down by glutathione beads and monitored by anti-HA antibody. Chemistry & Biology 2009 16, 644-656DOI: (10.1016/j.chembiol.2009.05.010) Copyright © 2009 Elsevier Ltd Terms and Conditions

Figure 7 Amitriptyline Prevents Neurons from Apoptosis in a TrkA-Dependent Manner (A) Amitriptyline activates TrkB independent of TrkA receptor. Cortical neurons (TrkA −/−) were pretreated with a variety of compounds for 30 min and the cell lysates were analyzed by immunoblotting with anti-p-TrkB and anti-p-TrkA. (B) Amitriptyline activates TrkA in TrkB null neurons. Cortical neurons from TrkB +/− × TrkB +/− mice were treated with amitriptyline or imipramine for 30 min. Amitriptyline but not imipramine activated TrkA receptor in both wild-type and TrkB knockout neurons (top and third panels). (C) Amitriptyline selectively activates TrkA F592A, which can be blocked by 1NMPP1. The primary cultures were pretreated for 30 min with either K252a (100 nM) or 1NMPP1 (100 nM), followed by 0.5 μM amitriptyline, imipramine, or fluoxetine for 30 min. Amitriptyline-provoked TrkA activation was selectively inhibited by 1NMPP1 but not by K252a (top). (D) Amitriptyline suppresses KA-induced neuronal cell death in TrkA F592A mutant mice, which can be blocked by 1NMPP1. TrkA F592A knockin mice were treated with the following reagents: saline, 1NMPP1, KA, 1NMPP1 + KA, amitriptyline, 1NMPP1 + amitriptyline, and 1NMPP1 + amitriptyline + KA, as described in the Experimental Procedures. Immunoblotting was conducted with the indicated antibodies. (E) TrkB activation is dispensable for the neuroprotective effect of amitriptyline. Chemistry & Biology 2009 16, 644-656DOI: (10.1016/j.chembiol.2009.05.010) Copyright © 2009 Elsevier Ltd Terms and Conditions